Methods For Enhancing The Degradation Or Conversion Of Cellulosic Material

ABSTRACT

The present invention relates to methods for degrading or converting a cellulosic material and for producing substances from the cellulosic material under high temperature conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/465,272, file on Mar. 21, 2017, which is a divisional applicationof U.S. application Ser. No. 14/112,023, filed on Dec. 17, 2013, whichis a 35 U.S.C. 371 national application of PCT/US2012/035486 filed onApr. 27, 2012, which claims priority or the benefit under 35 U.S.C. 119of U.S. Provisional. Application No. 61/480,860 filed on Apr. 29, 2011,the contents of which are fully incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made in part with Government support underCooperative Agreement DE-FC36-08GO18080 awarded by the Department ofEnergy. The government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods for degrading or converting acellulosic material and for producing substances from the cellulosicmaterial.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose isolatedGH61 polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Aspergillus fumigatus. WO 2011/005867discloses isolated GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Penicillium pinophilum. WO2008/151043 discloses methods of increasing the activity of a GH61polypeptide having cellulolytic enhancing activity by adding a solubleactivating divalent metal cation to a composition comprising thepolypeptide.

Banergee et al., 2010, Bioresource Technology 101: 9097-9105, disclosesynthetic multi-component enzyme mixtures for deconstruction oflignocellulosic biomass at 50° C. Viikari et al., 2007, Adv. Biochem.Engin./Biotechnol. 108: 121-145, disclose thermostable enzymes inlignocellulose hydrolysis.

There is a need in the art for new enzyme compositions to increaseefficiency and to provide cost-effective enzyme solutions for hightemperature saccharification of cellulosic material.

The present invention provides isolated polypeptides having cellulolyticenhancing activity and isolated nucleic acid sequences encoding thepolypeptides to improve the conversion of cellulosic feedstocks athigher temperatures.

SUMMARY OF THE INVENTION

The present invention relates to methods for degrading or converting acellulosic material, comprising: treating the cellulosic material underhigh temperature conditions with an enzyme composition in the presenceof a GH61 polypeptide having cellulolytic enhancing activity selectedfrom the group consisting of:

(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least medium-high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a GH61 polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial under high temperature conditions with an enzyme composition inthe presence of a GH61 polypeptide having cellulolytic enhancingactivity; (b) fermenting the saccharified cellulosic material with oneor more (e.g., several) fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation; wherein the GH61 polypeptide having cellulolyticenhancing activity is selected from the group consisting of:

(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least medium-high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a GH61 polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified under high temperature conditions with anenzyme composition in the presence of a GH61 polypeptide havingcellulolytic enhancing activity selected from the group consisting of:

(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least medium-high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a GH61 polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence (SEQ ID NO: 1) and deduced aminoacid sequence (SEQ ID NO: 2) of a Trichoderma reesei RutC30 geneencoding a GH61A polypeptide having cellulolytic enhancing activity. Thesignal sequence is underlined and the intronic sequence is in italics.

FIG. 2 shows a restriction map of pAJ223.

FIG. 3 shows the effect of GH61 polypeptides having cellulolyticenhancing activity on PCS-hydrolyzing activity of a Trichodermareesei-based cellulase composition and a high-temperature enzymecomposition.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain (Teeri, 1997, Crystalline cellulosedegradation: New insight into the function of cellobiohydrolases, Trendsin Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reeseicellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determinedaccording to the procedures described by Lever et al., 1972, Anal.Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288;and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the presentinvention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., 60° C., or 65° C., compared to a controlhydrolysis without addition of cellulolytic enzyme protein. Typicalconditions are 1 ml reactions, washed or unwashed PCS, 5% insolublesolids, 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., 60° C.,or 65° C., 72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzesendohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulosederivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.Fragment: The term “fragment” means a polypeptide or a domain thereofhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or a domain thereof;wherein the fragment has cellulolytic enhancing activity or cellulosebinding activity. In one aspect, a fragment contains at least 280 aminoacid residues, e.g., at least 295 amino acid residues or at least 310amino acid residues of the mature polypeptide of SEQ ID NO: 2.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., 60° C., or 65° C.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more (e.g., several) or all of the naturally occurringconstituents with which it is associated in nature; (3) any substancemodified by the hand of man relative to that substance found in nature;or (4) any substance modified by increasing the amount of the substancerelative to other components with which it is naturally associated(e.g., recombinant production in a host cell; multiple copies of a geneencoding the substance; and use of a stronger promoter than the promoternaturally associated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 22 to 344 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 21 of SEQ ID NO: 2 are a signal peptide. It isknown in the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide. It is alsoknown in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature GH61 polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 64 to 1086 of SEQ ID NO: 1 orthe cDNA sequence thereof based on the SignalP program (Nielsen et al.,1997, supra) that predicts nucleotides 1 to 63 of SEQ ID NO: 1 encode asignal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C.,compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsaevrd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellulolytic enhancingactivity of the mature polypeptide of SEQ ID NO: 2.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellulolytic enhancing activity. In one aspect, asubsequence contains at least 840 nucleotides, e.g., at leastnucleotides 885 or at least nucleotides 930 of SEQ ID NO: 1; or the cDNAsequence thereof.

Variant: The term “variant” means a GH61 polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for degrading or converting acellulosic material, comprising: treating the cellulosic material underhigh temperature conditions with an enzyme composition in the presenceof a GH61 polypeptide having cellulolytic enhancing activity selectedfrom the group consisting of:

(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least medium-high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a GH61 polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial under high temperature conditions with an enzyme composition inthe presence of a GH61 polypeptide having cellulolytic enhancingactivity; (b) fermenting the saccharified cellulosic material with oneor more (e.g., several) fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation; wherein the GH61 polypeptide having cellulolyticenhancing activity is selected from the group consisting of:

(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least medium-high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a GH61 polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition under hightemperature conditions in the presence of a GH61 polypeptide havingcellulolytic enhancing activity selected from the group consisting of:

(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2;

(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least medium-high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii);

(c) a GH61 polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The methods of the present invention can be used to saccharify acellulosic material under high temperature conditions to fermentablesugars and to convert the fermentable sugars to many useful fermentationproducts, e.g., fuel, potable ethanol, and/or platform chemicals (e.g.,acids, alcohols, ketones, gases, and the like). The ability to performthe saccharification in the presence of a GH61 polypeptide havingcellulolytic enhancing activity under higher temperature conditions thancustomarily used, e.g., 50° C., provides several advantages.Non-limiting examples of such advantages include an increase inhydrolytic efficiency of the enzymes, reduction of viscosity of thecellulosic material, reduced risk of microbial contamination, andcost-effectiveness of degrading cellulosic material.

The high temperature conditions are preferably a temperature of about54° C. to about 70° C. for about 6 to about 168 hours at a pH of about 3to about 8 and a dry solids content of a cellulosic material of about 5to about 50 wt %.

In one aspect of the high temperature conditions, the temperature is inthe range of about 54° C. to about 70° C. In another aspect, thetemperature is in the range of about 54° C. to about 65° C. In anotheraspect, the temperature is in the range of about 55° C. to about 65° C.In another aspect, the temperature is in the range of about 56° C. toabout 65° C. In another aspect, the temperature is in the range of about54° C. to about 60° C. In another aspect, the temperature is in therange of about 55° C. to about 60° C. In another aspect, the temperatureis in the range of about 56° C. to about 60° C. In another aspect, thetemperature is about 54° C., about 55° C., about 56° C., about 57° C.,about 58° C., about 59° C., about 60° C., about 61° C., about 62° C.,about 63° C., about 64° C., about 65° C., about 66° C., about 67° C.,about 68° C., about 69° C., or about 70° C. In another aspect, thetemperature is at least 54° C., at least 55° C., at least 56° C., atleast 57° C., at least 58° C., at least 59° C., at least 60° C., atleast 61° C., at least 62° C., at least 63° C., at least 64° C., atleast 65° C., at least 66° C., at least 67° C., at least 68° C., atleast 69° C., or at least 70° C.

In each of the aspects above for the temperature of the high temperatureconditions, the saccharification is performed for about 6 to about 168hours, about 6 to about 144 hours, about 6 to about 120 hours, about 6to about 96 hours, about 6 to about 72 hours, about 6 to about 48 hours,about 6 to about 24 hours, about 6 to about 12 hours, about 6 hours,about 12 to about 168 hours, about 12 to about 144 hours, about 12 toabout 120 hours, about 12 to about 96 hours, about 12 to about 72 hours,about 12 to about 48 hours, about 12 to about 24 hours, about 12 hours,about 24 to about 168 hours, about 24 to about 144 hours, about 24 toabout 120 hours, about 24 to about 96 hours, about 24 to about 72 hours,about 24 to about 48 hours, about 24 hours, about 48 to about 168 hours,about 48 to about 144 hours, about 48 to about 120 hours, about 48 toabout 96 hours, about 48 to about 72 hours, about 48 hours, about 72 toabout 168 hours, about 72 to about 144 hours, about 72 to about 120hours, about 72 to about 96 hours, about 72 hours, about 96 to about 168hours, about 96 to about 144 hours, about 96 to about 120 hours, about96 hours, about 120 to about 168 hours, about 120 to about 144 hours,about 120 hours, about 144 to about 168 hours, about 144 hours, or about168 hours. In each of the aspects above for the temperature of the hightemperature conditions, the saccharification is performed for at least 6hours, at least 12 hours, at least 24 hours, at least 48 hours, at least72 hours, at least 96 hours, at least 120 hours, at least 144 hours, orat least 168 hours.

In each of the aspects above for the temperature and thesaccharification time of the high temperature conditions, thesaccharification is performed at a pH of about 3 to about 8, about 3.5to about 7.5, about 4 to about 7, about 4 to about 6.5, about 4.5 toabout 6.5, about 4.5 to about 6, about 4 to about 6, about 5 to about 6,about 4.5 to about 5.5, or about 5 to about 5.5.

In each of the aspects above for the temperature, the saccharificationtime, and the saccharification pH of the high temperature conditions,the dry solids content of the cellulosic material is about 5 to about 50wt %, about 10 to about 40 wt %, about 15 to about 30 wt %, about 20 toabout 30 wt %, or about 25 to about 30 wt %. In each of the aspectsabove for the temperature, the saccharification time, and thesaccharification pH of the high temperature conditions, the dry solidscontent of the cellulosic material is at least 5 wt %, at least 10 wt %,at least 15 wt %, at least 20 wt %, at least 25 wt %, at least 30 wt %,at least 35 wt %, at least 40 wt %, or at least 45 wt %.

In one preferred embodiment, the high temperature conditions are atemperature of about 54° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 55° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 56° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 57° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 58° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 59° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 60° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 61° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 62° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 63° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 64° C. to about 65° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 54° C. to about 64° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 54° C. to about 63° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 54° C. to about 62° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 54° C. to about 61° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 54° C. to about 60° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 55° C. to about 64° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 56° C. to about 64° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 56° C. to about 63° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 57° C. to about 62° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 58° C. to about 61° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

In another preferred embodiment, the high temperature conditions are atemperature of about 59° C. to about 60° C. for about 48 to about 72hours at a pH of about 4 to about 6 and a dry solids content of thecellulosic material of about 15 to about 30 wt %.

Polypeptides Having Cellulolytic Enhancing Activity and PolynucleotidesThereof

In an embodiment, the isolated GH61 polypeptides having cellulolyticenhancing activity have a sequence identity to the mature polypeptide ofSEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%; which have cellulolytic enhancing activity. In one aspect,the GH61 polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

A GH61 polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 2 or anallelic variant thereof; or is a fragment thereof having cellulolyticenhancing activity. In another aspect, the GH61 polypeptide havingcellulolytic enhancing activity comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the GH61 polypeptidehaving cellulolytic enhancing activity comprises or consists of aminoacids 22 to 344 of SEQ ID NO: 2.

In another embodiment, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.)

The polynucleotide of SEQ ID NO: 1, or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2, or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having cellulolytic enhancing activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic DNA or cDNA of a cell of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a GH61 polypeptide having cellulolytic enhancing activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the cDNA sequence thereof; (iv) the full-lengthcomplement thereof; or (v) a subsequence thereof; under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe GH61 polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; ora fragment thereof. In another aspect, the nucleic acid probe is SEQ IDNO: 1, the mature polypeptide coding sequence of SEQ ID NO: 1, or thecDNA sequence thereof.

In another embodiment, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, or the cDNA sequence thereof, of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%.

In another embodiment, the isolated GH61 polypeptides havingcellulolytic enhancing activity are variants of the mature polypeptideof SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion atone or more (e.g., several) positions. In one aspect, the number ofamino acid substitutions, deletions and/or insertions introduced intothe mature polypeptide of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, thatis conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, thermal activity of the polypeptide, alter the substratespecificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A GH61 polypeptide having cellulolytic enhancing activity may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a Gram-positive bacterial polypeptide such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces polypeptide having [enzyme] activity, or a Gram-negativebacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium,Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,Salmonella, or Ureaplasma polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aurantiporus, Aureobasidium, Botryosphaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylariapolypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

In another aspect, the polypeptide is a Trichoderma polypeptide. Inanother aspect, the polypeptide is a Trichoderma reesei polypeptide. Inanother aspect, the polypeptide is a Trichoderma reesei RutC30polypeptide.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

Polynucleotides encoding GH61 polypeptides having cellulolytic enhancingactivity can be isolated and utilized to practice the methods of thepresent invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Trichoderma, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not result in a change in the aminoacid sequence of the polypeptide, but which correspond to the codonusage of the host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions that may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

Nucleic Acid Constructs

A polynucleotide encoding a GH61 polypeptide having cellulolyticenhancing activity may be operably linked to one or more controlsequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus nidulans acetamidase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase, Fusariumoxysporum trypsin-like protease (WO 96/00787), Fusarium venenatumamyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900),Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase,Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus nigerglucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

A polynucleotide encoding a GH61 polypeptide and various nucleic acidsand control sequences described herein may be joined together to producea recombinant expression vector that may include one or more convenientrestriction sites to allow for insertion or substitution of thepolynucleotide at such sites. Alternatively, the polynucleotide may beexpressed by inserting the polynucleotide or a nucleic acid constructcomprising the polynucleotide into an appropriate vector for expression.In creating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Recombinant host cells comprising a polynucleotide encoding a GH61polypeptide having cellulolytic enhancing activity operably linked toone or more control sequences that direct the production of apolypeptide can be advantageously used in the recombinant production ofthe polypeptide. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

A GH61 polypeptide of the present invention can be produced usingmethods comprising: (a) cultivating a cell, which in its wild-type formproduces the polypeptide, under conditions conducive for production ofthe polypeptide; and optionally (b) recovering the polypeptide. In oneaspect, the cell is a Trichoderma cell. In another aspect, the cell is aTrichoderma reesei cell. In another aspect, the cell is Trichodermareesei RutC30.

A GH61 polypeptide of the present invention can also be produced usingmethods comprising: (a) cultivating a recombinant host cell of thepresent invention under conditions conducive for production of thepolypeptide; and optionally (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a GH61 polypeptide of the presentinvention. The fermentation broth product further comprises additionalingredients used in the fermentation process, such as, for example,cells (including, the host cells containing the gene encoding thepolypeptide of the present invention which are used to produce thepolypeptide of interest), cell debris, biomass, fermentation mediaand/or fermentation products. In some embodiments, the composition is acell-killed whole broth containing organic acid(s), killed cells and/orcell debris, and culture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising a GH61polypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a GH61 polypeptide of the presentinvention as the major enzymatic component, e.g., a mono-componentcomposition. Alternatively, the compositions may comprise multipleenzymatic activities, such as one or more (e.g., several) enzymesselected from the group consisting of a cellulase, a hemicellulase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin. The compositions may alsocomprise one or more (e.g., several) enzymes selected from the groupconsisting of a hydrolase, an isomerase, a ligase, a lyase, anoxidoreductase, or a transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Processing of Cellulosic Material

The processing of a cellulosic material according to the methods of thepresent invention can be accomplished using processes conventional inthe art. Moreover, the methods of the present invention can beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention. The productionof a desired fermentation product from the cellulosic material typicallyinvolves pretreatment, enzymatic hydrolysis (saccharification), andfermentation.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition in the presence of a GH61 polypeptide having cellulolyticenhancing activity of the present invention. The enzymes of thecompositions can be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 6 or about 12 to about 120 hours or about 168 hours, e.g., about24 to about 168 hours, about 16 to about 72 hours, about 48 to about 72hours, or about 24 to about 48 hours. The temperature can be in therange of about 25° C. to about 70° C., but preferably in a the range ofabout 54° C. to about 70° C. as defined herein. The pH is in the rangeof preferably about 3 to about 8, e.g., about 3.5 to about 7, about 4 toabout 6, about 4.5 to about 5.5, or about 5.0 to about 5.5. The drysolids content is in the range of preferably about 5 to about 50 wt %,e.g., about 10 to about 40 wt %, about 15 to about 30 wt %, or about 20to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading orconverting the cellulosic material under the high temperature conditionsdescribed herein.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin. In another aspect, the cellulase is preferably one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the hemicellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an acetylmannan esterase,an acetylxylan esterase, an arabinanase, an arabinofuranosidase, acoumaric acid esterase, a feruloyl esterase, a galactosidase, aglucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, axylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises anendoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises a cellobiohydrolase anda beta-glucosidase. In another aspect, the enzyme composition comprisesan endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes enzymes and/orhemicellulolytic enzymes, the cellulosic material, the concentration ofcellulosic material, the pretreatment(s) of the cellulosic material,temperature, time, pH, and inclusion of fermenting organism (e.g., yeastfor Simultaneous Saccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a GH61 polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a GH61 polypeptide havingcellulolytic enhancing activity to cellulolytic or hemicellulolyticenzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 toabout 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g perg of cellulolytic or hemicellulolytic enzyme.

In the methods of the present invention, a GH61 polypeptide havingcellulolytic enhancing activity of the present invention is used in thepresence of a soluble activating divalent metal cation according to WO2008/151043, e.g., manganese sulfate.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more (e.g., several) nitrogen atoms. In one aspect, thenitrogen-containing compound comprises an amine, imine, hydroxylamine,or nitroxide moiety. Non-limiting examples of the nitrogen-containingcompounds include acetone oxime; violuric acid; pyridine-2-aldoxime;2-aminophenol; 1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more (e.g., several) sulfur atoms. In one aspect, thesulfur-containing comprises a moiety selected from thionyl, thioether,sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonicester. Non-limiting examples of the sulfur-containing compounds includeethanethiol; 2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonicacid; benzenethiol; benzene-1,2-dithiol; cysteine; methionine;glutathione; cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ toabout 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material (collectively hereinafter“polypeptides having enzyme activity”) can be derived or obtained fromany suitable origin, including, bacterial, fungal, yeast, plant, ormammalian origin. The term “obtained” also means herein that the enzymemay have been produced recombinantly in a host organism employingmethods described herein, wherein the recombinantly produced enzyme iseither native or foreign to the host organism or has a modified aminoacid sequence, e.g., having one or more (e.g., several) amino acids thatare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme that is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained recombinantly, such as by site-directed mutagenesis orshuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™7069 W (Röhm GmbH), FIBREZYME® LDI (Dyadic International, Inc.),FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150 L (DyadicInternational, Inc.). The cellulase enzymes are added in amountseffective from about 0.001 to about 5.0 wt % of solids, e.g., about0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % ofsolids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thermoascus aurantiacus endoglucanase II,Thielavia terrestris NRRL 8126 CEL6B endoglucanase, Thielavia terrestrisNRRL 8126 CEL6C endoglucanase, Thielavia terrestris NRRL 8126 CEL7Cendoglucanase, Thielavia terrestris NRRL 8126 CEL7E endoglucanase,Thielavia terrestris NRRL 8126 CEL7F endoglucanase, Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase, and Trichoderma reeseistrain No. VTT-D-80133 endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Aspergillus fumigatus cellobiohydrolase II, Chaetomiumthermophilum cellobiohydrolase I, Chaetomium thermophilumcellobiohydrolase II, Humicola insolens cellobiohydrolase I,Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium emersonii cellobiohydrolase I, Thielavia hyrcaniecellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740 L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

In a preferred embodiment, the enzyme composition is a high temperaturecomposition, i.e., a composition that is able to hydrolyze a cellulosicmaterial in the range of about 54° C. to about 70° C. In anotherpreferred embodiment, the enzyme composition is a high temperaturecomposition, i.e., a composition that is able to hydrolyze a cellulosicmaterial at a temperature of about 54° C., about 55° C., about 56° C.,about 57° C., about 58° C., about 59° C., about 60° C., about 61° C.,about 62° C., about 63° C., about 64° C., about 65° C., about 66° C.,about 67° C., about 68° C., about 69° C., or about 70° C. In anotherpreferred embodiment, the enzyme composition is a high temperaturecomposition, i.e., a composition that is able to hydrolyze a cellulosicmaterial at a temperature of at least 54° C., at least 55° C., at least56° C., at least 57° C., at least 58° C., at least 59° C., at least 60°C., at least 61° C., at least 62° C., at least 63° C., at least 64° C.,at least 65° C., at least 66° C., at least 67° C., at least 68° C., atleast 69° C., or at least 70° C.

In another preferred embodiment, the enzyme composition is a hightemperature composition as disclosed in PCT/US2010/055723 (WO2011/057140), which is incorporated herein in its entirety by reference.

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art.

Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, and/or oligosaccharides, directly or indirectly into thedesired fermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In another more preferred aspect, the yeast is Saccharomyces cerevisiae.In another more preferred aspect, the yeast is Saccharomyces distaticus.In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more (e.g., several) hydroxyl moieties. In a morepreferred aspect, the alcohol is n-butanol. In another more preferredaspect, the alcohol is isobutanol. In another more preferred aspect, thealcohol is ethanol. In another more preferred aspect, the alcohol ismethanol. In another more preferred aspect, the alcohol is arabinitol.In another more preferred aspect, the alcohol is butanediol. In anothermore preferred aspect, the alcohol is ethylene glycol. In another morepreferred aspect, the alcohol is glycerin. In another more preferredaspect, the alcohol is glycerol. In another more preferred aspect, thealcohol is 1,3-propanediol. In another more preferred aspect, thealcohol is sorbitol. In another more preferred aspect, the alcohol isxylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R.,2002, The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more (e.g., several) ketone moieties. In another morepreferred aspect, the ketone is acetone. See, for example, Qureshi andBlaschek, 2003, supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Detergent Compositions

A GH61 polypeptide having cellulolytic enhancing activity of the presentinvention may be added to and thus become a component of a detergentcomposition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

In a specific aspect, the present invention provides a detergentadditive comprising a GH61 polypeptide having cellulolytic enhancingactivity as described herein. The detergent additive as well as thedetergent composition may comprise one or more (e.g., several) enzymessuch as a protease, lipase, cutinase, an amylase, carbohydrase,cellulase, pectinase, mannanase, arabinase, galactanase, xylanase,oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include CELLUZYME™ and CAREZYME™(Novozymes A/S), CLAZINASE™, and PURADAX HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metalloprotease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more (e.g., several) of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235, and 274.

Preferred commercially available protease enzymes include ALCALASE™SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novozymes A/S),MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™,LIPEX™, and LIPOLASE ULTRA™ (Novozymes A/S).

Amylases:

Suitable amylases (alpha and/or beta) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g., a special strain of Bacillus licheniformis, described inmore detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more (e.g., several) of the following positions:15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208,209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available amylases are DURAMYL™, TERMAMYL™, FUNGAMYL™ andBAN™ (Novozymes A/S), and RAPIDASE™ and PURASTAR™ (from GenencorInternational Inc.).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more (e.g., several)enzymes, or by adding a combined additive comprising all of theseenzymes. A detergent additive of the invention, i.e., a separateadditive or a combined additive, can be formulated, for example, as agranulate, liquid, slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

A detergent composition of the present invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more (e.g., several)surfactants, which may be non-ionic including semi-polar and/or anionicand/or cationic and/or zwitterionic. The surfactants are typicallypresent at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more (e.g., several) polymers.Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),poly(vinylimidazole), polycarboxylates such as polyacrylates,maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acidcopolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

In the detergent compositions, a GH61 polypeptide having cellulolyticenhancing activity may be added in an amount corresponding to 0.001-100mg of protein, preferably 0.005-50 mg of protein, more preferably0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, mostpreferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg ofprotein per liter of wash liquor.

A polypeptide of the invention having cellulolytic enhancing activitymay also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Media

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

M400 medium was composed of 50 g of maltodextrin, 2 g of MgSO₄.7H₂O, 2 gof KH₂PO₄, 4 g of citric acid, 8 g of yeast extract, 2 g of urea, 0.5 mlof AMG trace metals solution, 0.5 g CaCl₂, and deionized water to 1liter.

AMG trace metals solution was composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 3 g of citric acid, and deionized water to 1 liter.

Minimal medium plates were composed of 6 g of NaNO₃, 0.52 g of KCl, 1.52g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g of Noble agar,20 ml of 50% glucose, 2.5 ml of a 20% MgSO₄.7H₂O solution, 20 ml of a0.02% biotin solution, and deionized water to 1 liter.

COVE plates were composed of 218 g of sorbitol, 20 g of agar, 20 ml ofCOVE salts solution, 10 mM acetamide, 15 mM CsCl, and deionized water to1 liter. The solution was adjusted to pH 7.0 before autoclaving.

COVE salts solution was composed of 26 g of KCl, 26 g of MgSO₄.7H₂O, 76g of KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to1 liter.

COVE trace elements solution was composed of 0.04 g of Na₂B₄O₇.10H₂O,0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO4.7H₂O, 0.7 g of MnSo₄.H₂O, 0.8 g ofNa₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

YPM medium was composed of 1% yeast extract, 2% peptone, andfilter-sterilized 2% maltodextrin added after autoclaving.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, and deionized water to 1 liter.

MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g ofNaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeast extract, 2 g of urea,0.5 ml of AMG trace metals solution, and deionized water to 1 liter; pH5.0.

200×AMG trace metals solution was composed of 3 g of citric acid, 14.3 gof ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.H₂O, and deionized water to 1 liter.

2XYT plates were composed of 16 g of tryptone, 10 g of yeast extract, 5g of NaCl, 15 g of Noble agar, and deionized water to 1 liter.

MY25 medium was composed of 25 g of maltodextrin, 2 g of MgSO₄.7H₂O, 10g of KH₂PO₄, 2 g of citric acid, 2 g of K₂SO₄, 2 g of urea, 10 g ofyeast extract, 5 ml of AMG trace metals solution, and deionized water to1 liter, adjusted to pH 6.

SY50 medium was composed of 50 g of sucrose, 2 g of MgSO₄.7H₂O, 10 g ofKH₂PO₄, anhydrous, 2 g of K₂SO₄, 2 g of citric acid, 10 g of yeastextract, 2 g of urea, 0.5 g of CaCl₂.2H₂O, 0.5 g of 200×AMG trace metalssolution, and deionized water to 1 liter, pH 6.0.

Example 1: Cloning and Expression of the Trichoderma reesei RutC30 GH61APolypeptide Genomic Sequence

The Trichoderma reesei GH61A polypeptide coding sequence was amplifiedby PCR from T. reesei RutC30 genomic DNA using the primers shown below.Genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc.,Valencia, Calif., USA).

Primer TrGH61_infus.1S: (SEQ ID NO: 3)5′-caactggatttaccatgatccagaagctttcc-3′ Primer TrGH61_infus.1A:(SEQ ID NO: 4) 5′-cagtcacctctagttaattaactagttaaggcactgggc-3′

The amplification reaction (50 μl) was composed of a 10 mM blend ofdATP, dTTP, dGTP, and dCTP, 275 ng of T. reesei RutC30 genomic DNA, 50pmole of primer TrGH61_infus.1S, 50 pmole of primer TrGH61_infus.1A, and10 μl of 5× PHUSION™ PCR Master Mix (New England Biolabs, Ipswich,Mass., USA). The amplification reaction was incubated in an EPPENDORF®MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA)programmed for 1 cycle at 98° C. for 1 minute; 30 cycles each at 98° C.for 15 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute; and afinal extension step at 72° C. for 5 minutes.

A 1124 bp PCR reaction product was visualized by 1% agarose gelelectrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodiumEDTA (TAE) buffer. The 1070 bp PCR product was digested with Dpn I andthen purified using a NUCLEOSPIN® Extract II column (Clontech, MountainView, Calif., USA) according to the manufacturer's instructions.

The vector pAILo2 (WO 2004/099228) was linearized by digestion with NcoI and Pac I. The digested fragment was isolated by 1% agarose gelelectrophoresis using TAE buffer, excised from the gel, and purifiedusing a GFX® PCR DNA and Gel Band Purification Kit (GE Healthcare,Piscataway, N.J., USA). Cloning of the purified PCR fragment into thepurified linearized pAILo2 vector was performed with an IN-FUSION™Cloning Kit (Clontech Laboratories, Inc, Mountain View, Calif., USA).The reaction (10 μl) was composed of 2 μl of 1× IN-FUSION™ Buffer(Clontech Laboratories, Inc, Mountain View, Calif., USA), 1 μl ofIN-FUSION™ enzyme (Clontech Laboratories, Inc, Mountain View, Calif.,USA) (diluted 1:10), 150 ng of pAILo2 digested with Nco I and Pac I, and150 ng of the T. reesei 1070 bp PCR product. The final plasmid productfrom the IN-FUSION™ reaction was designed so as to recreate the Pac Irestriction site at the 3′ end of the coding sequence upon properinsertion of the PCR fragment into pAILo2, whereas at the 5′ end of thecoding sequence the Nco I restriction site was not preserved. After thereaction was incubated at 37° C. for 15 minutes and then at 50° C. for15 minutes, 40 μl of 0.1 mM EDTA-10 mM Tris (TE) buffer was added. A 3μl sample of the reaction was used to transform E. coli XL10 GOLD®competent cells (Stratagene, Inc., Santa Clara, Calif., USA) accordingto the manufacturer's instructions. After a recovery period, twoaliquots of 100 μl and 300 μl from the transformation reaction werespread onto 150 mm 2XYT plates supplemented with 100 μg of ampicillinper ml. The plates were incubated overnight at 37° C. Plasmid DNA fromthe resulting colonies were prepared using a BIOROBOT® 9600 (QIAGENInc., Valencia, Calif., USA). Subcloned PCR inserts were sequenced usingan Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems,Foster City, Calif., USA). Sequence analysis from a single colonyplasmid confirmed the identity of the DNA sequence encoding theTrichoderma reesei GH61A polypeptide. This plasmid was designated pAJ223(FIG. 2).

Example 2: Characterization of the Trichoderma reesei RutC30 GH61APolypeptide Coding Sequence

The genomic DNA sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Trichoderma reesei RutC30 GH61A polypeptideencoding sequence are shown in FIG. 1. The genomic DNA sequence of 1089bp (including the stop codon) contains one intron located at nucleotides187 to 240 of SEQ ID NO: 1. The genomic DNA fragment encodes apolypeptide of 344 amino acids. The % G+C content of the maturepolypeptide coding sequence is 60%. Using the SignalP software program(Nielsen et al., 1997, Protein Engineering 10: 1-6), a signal peptide of21 residues was predicted. The SignalP prediction is in accord with thenecessity for having a histidine reside at the N-terminus in order forproper metal binding and hence protein function to occur (See Harris etal., 2010, Biochemistry 49: 3305, and Quinlan et al., 2011, Proc. Natl.Acad. Sci. USA 108: 15079). The predicted mature protein contains 323amino acids with a predicted molecular mass of 33.4 kDa and a predictedisoelectric point of 5.09.

Example 3: Heterologous Expression of the Trichoderma reesei GH61APolypeptide in Aspergillus oryzae

Aspergillus oryzae JaL250 (WO 99/61651) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422, and transformed with 5 μg of pAJ223. Twenty-fourtransformants were isolated to individual PDA plates. Confluent PDAplates of the transformants were washed with 8 ml of 0.01% TWEEN® 20 tocreate spore stocks. Twenty μl of each spore stock were inoculatedseparately into wells of a 24-well plate. Each well contained 1 ml ofM400 medium. The 24-well plate was incubated for 120 hours at 34° C.After incubation, a 12 μl sample of culture broth from each A. oryzaeJaL250 transformant was analyzed by SDS-PAGE using an 8-16% Tris-glycineSDS-PAGE gel (Bio-Rad Laboratories, Hercules, Calif., USA). A single A.oryzae transformant was identified by the presence of an approximately55 kDa band and designated A. oryzae AJ223-16.

A culture of A. oryzae AJ223-16 was cultivated in a 2800 Fernbach flaskcontaining 500 ml of M400 medium inoculated with 200 μl of a spore stockof A. oryzae AJ223-16. The Fernbach flask culture was grown at 34° C.with shaking at 250 rpm for 120 hours. The culture broth was thenfiltered using a 0.22 μm GP EXPRESS® PLUS Membrane filter (Millipore,Bedford, Mass., USA).

Example 4: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose, and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of the PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. Milled unwashed PCS (dry weight 32.35%) was preparedby milling whole slurry PCS in a Cosmos ICMG 40 wet multi-utilitygrinder (EssEmm Corporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plate wasthen sealed using an ALPS300™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at a specific temperature for72 hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.In case of unwashed PCS, the net concentrations ofenzymatically-produced sugars were determined by adjusting the measuredsugar concentrations for corresponding background sugar concentrationsin unwashed PCS at zero time point. All HPLC data processing wasperformed using MICROSOFT EXCEL™ software (Microsoft, Richland, Wash.,USA).

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=(glucose concentration/glucoseconcentration in a limit digest)×100. To calculate total conversion theglucose and cellobiose values were combined. Cellobiose concentrationwas multiplied by 1.053 in order to convert to glucose equivalents andadded to the glucose concentration. The degree of total celluloseconversion was calculated using the following equation: %conversion=([glucose concentration+1.053×(cellobioseconcentration)]/[(glucose concentration+1.053×(cellobiose concentration)in a limit digest])×100. The 1.053 factor for cellobiose takes intoaccount the increase in mass when cellobiose is converted to glucose. Inorder to calculate % conversion, a 100% conversion point was set basedon a cellulase control (100 mg of Trichoderma reesei cellulase per gramcellulose), and all values were divided by this number and thenmultiplied by 100. Triplicate data points were averaged and standarddeviation was calculated.

Example 5: Preparation of Penicillium emersonii Strain NN051602 GH7Cellobiohydrolase I

The Penicillium emersonii strain NN051602 Cel7 cellobiohydrolase I (SEQID NO: 5 [DNA sequence] and SEQ ID NO: 6 [deduced amino acid sequence])was obtained according to the procedure described below.

Penicillium emersonii was grown on a PDA plate at 45° C. for 3 days.Mycelia were collected directly from the plate into a sterilized mortarand frozen under liquid nitrogen. Frozen mycelia were ground, by mortarand pestle, to a fine powder, and genomic DNA was isolated using aDNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif., USA).

Oligonucleotide primers, shown below, were designed to amplify the GH7cellobiohydrolase I gene from genomic DNA of Penicillium emersonii. AnIN-FUSION™ CF Dry-down PCR Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Sense primer: (SEQ ID NO: 7)5′-ACACAACTGGGGATCCACCatgcttcgacgggctcttc-3′ Antisense primer:(SEQ ID NO: 8) 5′-GTCACCCTCTAGATCTCGCAGAGCAACTTCCGTCTACTTC-3′Bold letters represented the coding sequence (for the sense primer) orthe downstream sequence of the coding region (for the antisense primer).The remaining sequence was homologous to the insertion sites ofpPFJO355.

The expression vector pPFJO355 contains the Aspergillus oryzaeTAKA-amylase promoter, Aspergillus niger glucoamylase terminatorelements, pUC19 derived sequences for selection and propagation in E.coli, and an Aspergillus nidulans pyrG gene, which encodes an orotidinedecarboxylase for selection of a transformant of a pyrG mutantAspergillus strain.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium emersonii genomic DNA, 10 μl of 5×GCBuffer (Finnzymes, Espoo, Finland), 1.5 μl of DMSO, 2.5 mM each of dATP,dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™ High-Fidelity DNAPolymerase (Finnzymes, Espoo, Finland) in a final volume of 50 μl. Theamplification was performed using a Peltier Thermal Cycler (MJ ResearchInc., South San Francisco, Calif., USA) programmed for denaturing at 98°C. for 1 minute; 8 cycles of denaturing at 98° C. for 15 seconds,annealing at 65° C. for 30 seconds, with a 1° C. decrease per cycle, andelongation at 72° C. for 80 seconds; 23 cycles each at 98° C. for 15seconds, 66° C. for 30 seconds and 72° C. for 75 seconds; and a finalextension at 72° C. for 7 minutes. The heat block then went to a 4° C.soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where anapproximately 1.4 kb product band was excised from the gel, and purifiedusing an ILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit (GEHealthcare, Buckinghamshire, UK) according to the manufacturer'sinstructions.

Plasmid pPFJO355 was digested with Bam I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, excised from the gel, andpurified using an ILLUSTRA® GFX® PCR DNA and Gel Band Purification Kitaccording to the manufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning Kit resulting in pGH7ZY209383 inwhich transcription of the Penicillium emersonii GH7 cellobiohydrolase Igene was under the control of the Aspergillus oryzae TAKA alpha-amylasepromoter. In brief, 30 ng of pPFJO355 digested with Bam I and Bgl II,and 60 ng of the Penicillium emersonii GH7 cellobiohydrolase I PCRproduct were added to a reaction vial and resuspended in a final volumeof 10 μl by the addition of deionized water. The reaction was incubatedat 37° C. for 15 minutes and then 50° C. for 15 minutes. Five μl of thereaction were used to transform E. coli TOP10 competent cells(Invitrogen Corp., Carlsbad, Calif., USA). An E. coli transformantcontaining pGH7ZY209383 was detected by colony PCR and plasmid DNA wasprepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA). The Penicillium emersonii GH7 cellobiohydrolase I geneinsert in pGH7ZY209383 was confirmed by DNA sequencing using a 3730XLDNA Analyzer (Applied Biosystems Inc, Foster City, Calif., USA).

Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pGH7ZY209383. The transformationyielded about 50 transformants. Twelve transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C. with agitation at 150 rpm. After3 days incubation, 20 μl of supernatant from each culture were analyzedby SDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel (InvitrogenCorporation, Carlsbad, Calif., USA) with MES buffer according to themanufacturer's instructions. The resulting gel was stained with INSTANT®Blue (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of thecultures showed that the majority of the transformants had a majorsmeary band of approximately 50 kDa. The expression strain wasdesignated A. oryzae EXP03477.

Slants of A. oryzae EXP03477 were washed with 10 ml of YPM medium andinoculated into several 2 liter flasks containing 400 ml of YPM mediumto generate broth for characterization of the enzyme. The cultures wereharvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

A 1600 ml volume of the filtered broth of A. oryzae EXP03477 wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml of 25 mM Bis-Tris pH 6.5 buffer, dialyzed against the same buffer,and filtered through a 0.45 μm filter. The final volume was 200 ml. Thesolution was applied to a 40 ml Q SEPHAROSE® Fast Flow column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 25 mM Bis-Tris pH6.5, and the proteins were eluted with a linear NaCl gradient (0-0.4 M).Fractions with activity against phosphoric acid swollen cellulose (PASC)were collected and applied to a 40 ml PHENYL SEPHAROSE™ HIC column (GEHealthcare, Piscataway, N.J., USA) equilibrated in 20 mM PBS with 1.8 M(NH₄)₂SO₄ pH 7 buffer, and the proteins were eluted with 20 mM PBS pH 7.Fractions from the column with activity toward PASC as substrate wereanalyzed by SDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MESbuffer. Fractions with the correct molecular weight were pooled. Thenthe pooled solution was concentrated by ultrafiltration. Theconcentrated protein was dialyzed in 10 mM sodium acetate pH 5.0 using10 kDa MWCO Slide-A-Lyzer Dialysis Cassette (Thermo Fischer Scientific,Waltham, Mass., USA). Protein concentration was determined using aMicroplate BCA™ Protein Assay Kit (Thermo Fischer Scientific, Waltham,Mass., USA) in which bovine serum albumin was used as a proteinstandard.

Example 6: Preparation of Aspergillus fumigatus Cellobiohydrolase II

Aspergillus fumigatus NN055679 GH6A cellobiohydrolase II (CBHII) (SEQ IDNO: 9 [DNA sequence] and SEQ ID NO: 10 [deduced amino acid sequence])was prepared according to the following procedure.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the full-length open reading frame of the Aspergillus fumigatuscellobiohydrolase II gene from genomic DNA. A TOPO® Cloning Kit(Invitrogen Corp., Carlsbad, Calif., USA) was used to clone the PCRproduct. An IN-FUSION™ Cloning Kit was used to clone the fragment intopAILo2.

Forward primer: (SEQ ID NO: 11)5′-ACTGGATTTACCATGAAGCACCTTGCATCTTCCATCG-3′ Reverse primer:(SEQ ID NO: 12) 5′-TCACCTCTAGTTAATTAAAAGGACGGGTTAGCGT-3′Bold letters represent coding sequence. The remaining sequence containssequence identity compared with the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 500 ng of Aspergillus fumigatus genomic DNA, 1× ThermoPol Taqreaction buffer (New England Biolabs, Ipswich, Mass., USA), 6 μl of a 10mM blend of dATP, dTTP, dGTP, and dCTP, and 0.1 unit of Taq DNApolymerase (New England Biolabs, Ipswich, Mass., USA), in a final volumeof 50 μl. An EPPENDORF® MASTERCYCLER® 5333 was used to amplify thefragment programmed for 1 cycle at 98° C. for 2 minutes; and 35 cycleseach at 96° C. for 30 seconds, 61° C. for 30 seconds, and 72° C. for 2minutes. After the 35 cycles, the reaction was incubated at 72° C. for10 minutes and then cooled at 10° C. until further processed. To removethe A-tails produced by the Taq DNA polymerase the reaction wasincubated for 10 minutes at 68° C. in the presence of 1 unit of Pfx DNApolymerase (Invitrogen, Carlsbad, Calif., USA).

A 1.3 kb PCR reaction product was isolated by 0.8% GTG-agarose gelelectrophoresis (Cambrex Bioproducts, East Rutherford, N.J., USA) usingTAE buffer and 0.1 μg of ethidium bromide per ml. The DNA band wasvisualized with the aid of a DARK READER™ (Clare Chemical Research,Dolores, Colo.) to avoid UV-induced mutations. The 1.3 kb DNA band wasexcised with a disposable razor blade and purified using anULTRAFREE®-DA spin cup (Millipore, Billerica, Mass.) according to themanufacturer's instructions.

The purified 1.3 kb PCR product was cloned into pCR®4Blunt-TOPO®(Invitrogen, Carlsbad, Calif., USA). Two μl of the purified PCR productwere mixed with 1 μl of a 2 M sodium chloride and 1 μl of the vector.The reaction was incubated at room temperature for 15 minutes and then 2μl of the reaction were transformed into E. coli TOP10 competent cellsaccording to the manufacturer's instructions. Two aliquots of 100 μleach of the transformation reaction were spread onto two 150 mm 2XYTplates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C.

Eight recombinant colonies were used to inoculate liquid culturescontaining 3 ml of LB medium supplemented with 100 μg of ampicillin perml. Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600.Clones were analyzed by restriction digestion. Plasmid DNA from eachclone was digested with Eco RI and analyzed by agarose gelelectrophoresis as above. Six out of eight clones had the expectedrestriction digestion pattern and from these, clones 2, 4, 5, 6, 7 and 8were selected to be sequenced to confirm that there were no mutations inthe cloned insert. Sequence analysis of their 5-prime and 3-prime endsindicated that clones 2, 6 and 7 had the correct sequence. These threeclones were selected for re-cloning into pAILo2. One microliter aliquotof each clone was mixed with 17 μl of 10-fold diluted 0.1 M EDTA-10 mMTris (TE) and 1 μl of this mix was used to re-amplify the Aspergillusfumigatus GH6A coding region.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μl of the diluted mix of clones 2, 6 and 7, 1×PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of a 10mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNApolymerase (Invitrogen, Carlsbad, Calif., USA), and 1 μl of 50 mM MgSO₄,in a final volume of 50 μl. An EPPENDORF® MASTERCYCLER® 5333 was used toamplify the fragment programmed for 1 cycle at 98° C. for 2 minutes; and35 cycles each at 94° C. for 30 seconds, 61° C. for 30 seconds, and 68°C. for 1.5 minutes. After the 35 cycles, the reaction was incubated at68° C. for 10 minutes and then cooled to 10° C. until further processed.A 1.3 kb PCR reaction product was isolated by 0.8% GTG-agarose gelelectrophoresis using TAE buffer and 0.1 μg of ethidium bromide per ml.The DNA band was visualized with the aid of a DARKREADER™Transilluminator to avoid UV-induced mutations. The 1.0 kb DNA band wasexcised with a disposable razor blade and purified with an ULTRAFREE®-DAspin cup according to the manufacturer's instructions.

The vector pAILo2 was linearized by digestion with Nco I and Pac I. Thefragment was purified by gel electrophoresis and ultrafiltration asdescribed above. Cloning of the purified PCR fragment into thelinearized and purified pAILo2 vector was performed with an IN-FUSION™Cloning Kit. The reaction (20 μl) contained 2 μl of 1× IN-FUSION™Buffer, 1×BSA, 1 μl of IN-FUSION™ enzyme (diluted 1:10), 100 ng ofpAILo2 digested with Nco I and Pac I, and 50 ng of the Aspergillusfumigatus GH6A purified PCR product. The reaction was incubated at roomtemperature for 30 minutes. A 2 μl sample of the reaction was used totransform E. coli TOP10 competent cells according to the manufacturer'sinstructions. After a recovery period, two 100 μl aliquots from thetransformation reaction were plated onto 150 mm 2XYT plates supplementedwith 100 μg of ampicillin per ml. The plates were incubated overnight at37° C. A set of eight putative recombinant clones was selected at randomfrom the selection plates and plasmid DNA was prepared from each oneusing a BIOROBOT® 9600. Clones were analyzed by Pst I restrictiondigestion. Seven out of eight clones had the expected restrictiondigestion pattern. Clones 1, 2, and 3 were then sequenced to confirmthat there were no mutations in the cloned insert. Clone #2 was selectedand designated pAILo33.

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422, whichwere transformed with 5 μg of plasmid pAILo33. The transformationyielded about 30 transformants. Twenty-six transformants were isolatedto individual PDA plates.

Confluent PDA plates of four of the transformants were washed with 8 mlof 0.01% TWEEN® 20 and inoculated separately into 1 ml of MDU2BP mediumin sterile 24 well tissue culture plates and incubated at 34° C. Threedays after incubation, 20 μl of harvested broth from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that several transformants had a newmajor band of approximately 75 kDa. One transformant was designatedAspergillus oryzae JaL355 ALLO33 (EXP03191).

One hundred ml of shake flask medium were added to a 500 ml shake flask.The shake flask medium was composed of 50 g of sucrose, 10 g of KH₂PO₄,0.5 g of CaCl₂, 2 g of MgSO₄.7H₂O, 2 g of K₂SO₄, 2 g of urea, 10 g ofyeast extract, 2 g of citric acid, 0.5 ml of trace metals solution, anddeionized water to 1 liter. The trace metals solution was composed of13.8 g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄. H₂O, 2.5 gof CuSO₄.5H₂O, 3 g of citric acid, and deionized water to 1 liter. Theshake flask was inoculated with two plugs of Aspergillus oryzae JaL355ALLO33 (EXP03191) from a PDA plate and incubated at 34° C. on an orbitalshaker at 200 rpm for 24 hours. Fifty ml of the shake flask broth wasused to inoculate a 3 liter fermentation vessel.

A total of 1.8 liters of the fermentation batch medium was added to athree liter glass jacketed fermentor. The fermentation batch medium wascomposed per liter of 10 g of yeast extract, 24 g of sucrose, 5 g of(NH₄)₂SO₄, 2 g of KH₂PO₄, 0.5 g of CaCl₂.2H₂O, 2 g of MgSO₄.7H₂O, 1 g ofcitric acid, 2 g of K₂SO₄, 0.5 ml of anti-foam, and 0.5 ml of tracemetals solution. The trace metals solution was composed per liter of13.8 g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g ofCuSO₄.5H₂O, 3 g of citric acid, and deionized water to 1 liter.Fermentation feed medium was composed of maltose. The fermentation feedmedium was dosed at a rate of 0 to 4.4 g/l/hr for a period of 185 hours.The fermentation vessel was maintained at a temperature of 34° C. and pHwas controlled using an Applikon 1030 control system (ApplikonBiotechnology Inc., Foster City, Calif., USA) to a set-point of6.1+/−0.1. Air was added to the vessel at a rate of 1 vvm and the brothwas agitated by a Rushton impeller rotating at 1100 to 1300 rpm. At theend of the fermentation, whole broth was harvested from the vessel andcentrifuged at 3000×g to remove the biomass. The supernatant was sterilefiltered and stored at 5 to 10° C. The supernatant was filtered using a0.22 μm EXPRESS™ Plus Membrane (Millipore, Bedford, Mass., USA).

A 100 ml volume of filtered supernatant was buffer exchanged into 20 mMTris pH 8.0 using a 400 ml SEPHADEX™ G-25 column (GE Healthcare, UnitedKingdom) according to the manufacturer's instructions. The fractionswere pooled and adjusted to 1.2 M ammonium sulphate-20 mM Tris pH 8.0.The equilibrated protein was loaded onto a PHENYL SEPHAROSE™ 6 Fast Flowcolumn (high sub) (GE Healthcare, Piscataway, N.J., USA) equilibratedwith 20 mM Tris pH 8.0 with 1.2 M ammonium sulphate, and bound proteinswere eluted with 20 mM Tris pH 8.0 with no ammonium sulphate. Thefractions were pooled and protein concentration was determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 7: Preparation of Thermoascus aurantiacus CGMCC 0670 Cel5AEndoglucanase II

Thermoascus aurantiacus CGMCC 0670 cDNA encoding a Cel5A endoglucanaseII (SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acidsequence]) was cloned according to the following procedure. The T.aurantiacus strain was grown in 80 ml of CBH1 medium (2.5% AVICEL®, 0.5%glucose, 0.14% (NH₄)₂SO₄) in 500 ml Erlenmeyer baffled flasks at 45° C.for 3 days with shaking at 165 rpm. Mycelia were harvested bycentrifugation at 7000 rpm for 30 minutes and stored at −80° C. beforeuse for RNA extraction. RNA was isolated from 100 mg of mycelia using anRNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif., USA).

The cDNA for the Thermoascus aurantiacus endoglucanase was isolated byRT PCR using a 3′ RACE system and a 5′ RACE System (Invitrogen, LifeTechnologies, Carlsbad, Calif., USA) and primers BG025-1, BG025-2,BG025-3, and BG025-4 shown below to the N-terminal amino acids.

Primer BG025-1: (SEQ ID NO: 15)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC (T/C/A/G)GAATT-3′Primer BG025-2: (SEQ ID NO: 16)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC (T/C/A/G)GAGTT-3′Primer BG025-3: (SEQ ID NO: 17) 5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/C/A/G)GAATT-3′ Primer BG025-4: (SEQ ID NO: 18)5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC (T/C/A/G)GAGTT-3′

The RT PCR products were ligated into plasmid pGEM®-T using a pGEM®-TVector System (Promega, Madison, Wis., USA) and transformed into E. colistrain JM109 (New England Biolabs, Inc., Ipswich, Mass., USA). A singleclone harboring a plasmid containing the endoglucanase cDNA was isolatedand named pBGC1009.

PCR primers were designed to amplify the cDNA encoding the T aurantiacusendoglucanase from plasmid pBGC1009. Restriction enzyme sites Bsp HI andPac I were incorporated for in-frame cloning into the Aspergillus oryzaeexpression plasmid pBM120a (WO 2006/039541).

Primer 996261: (SEQ ID NO: 19) 5′-GATCTCATGAAGCTCGGCTCTCTCGT-3′       Bsp HI Primer 996167: (SEQ ID NO: 20)5′-TTAATTAATCAAAGATACGGAGTCAAAATAGG-3′    Pac I

The fragment of interest was amplified by PCR using an EXPAND™ HighFidelity PCR System (Roche Diagnostics, Mannheim, Germany). The PCRamplification reaction mixture contained 1 μl of 0.09 μg/μl pBGC1009, 1μl of primer 996261 (50 pmol/μl), 1 μl of primer 996167 (50 pmol/μl), 5μl of 10×PCR buffer (Roche Diagnostics, Mannheim, Germany) with 15 mMMgCl₂, 1 μl of dNTP mix (10 mM each), 37.25 μl of water, and 0.75 μl(3.5 U/μl) of DNA polymerase mix. An EPPENDORF® MASTERCYCLER®thermocycler was used to amplify the fragment programmed for 1 cycle at94° C. for 2 minutes; 10 cycles each at 94° C. for 15 seconds, 55° C.for 30 seconds, 72° C. for 1.5 minutes; 15 cycles each at 94° C. for 15seconds, 55° C. for 30 seconds, and 72° C. for 1.5 minutes plus a 5second elongation at each successive cycle; 1 cycle at 72° C. for 7minutes; and a 4° C. hold.

The 1008 bp PCR product was purified by 1% agarose gel electrophoresisusing TAE buffer, excised from the gel, and purified using a QIAQUICK®Gel Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purifiedproduct was ligated directly into pCR®2.1-TOPO® (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. The resultingplasmid was named pBM124a.

Plasmid pBM124a was digested with Bsp HI and Pac 1, purified by 1%agarose gel electrophoresis using TAE buffer, excised from the gel, andpurified using a QIAQUICK® Gel Purification Kit. The plasmid fragmentwas ligated to the vector pBM120a, which was digested with Nco I and PacI. The resulting expression plasmid was designated pBM123a. PlasmidpBM123a contains a duplicate NA2-tpi promoter driving expression of theThermoascus aurantiacus endoglucanase cDNA clone, the AMG terminator,and amdS as a selectable marker.

Aspergillus oryzae BECh2 (WO 2000/139322) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra, andtransformed with 6 μg of pBM123a. Primary transformants were selected onCOVE plates for 5 days. Transformants were spore purified twice prior toshake flask analysis.

Spores of the transformants were inoculated into 25 ml of MY25 medium in125 ml shake flasks. The cultures were incubated at 34° C., 200 rpm on aplatform shaker for five days. On day 3 and day 5, culture supernatantswere harvested and clarified by centrifugation to remove mycelia. Twentymicroliters of supernatant from three transformants were analyzed usinga CRITERION® stain-free, 10-20% gradient SDS-PAGE gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA) according to themanufacturer's instructions. SDS-PAGE profiles of the cultures showedthat all transformants had a new major band of approximately 32 kDa. Onetransformant was chosen and named A. oryzae EXP00858.

Plastic, non-baffled 500 ml shake flasks containing 100 ml of SY50medium were inoculated with 0.1 ml of a spore stock of A. oryzaeEXP00858, and incubated at 34° C., 200 rpm for 24 hours to produce aseed culture. Fifty ml of the seed culture were inoculated into a 2liter fermentation tank containing 2 liters of medium composed per literof 0.5 g of pluronic acid, 30 g of sucrose, 2 g of MgSO₄.7H₂O, 2 g ofanhydrous KH₂PO₄, 1 g of citric acid, 2 g of (NH₄)₂SO₄, 1 g of K₂SO₄, 20g of yeast extract, and 0.5 g of 200×AMG trace metals solution, pH 5.0.The fermentation was fed with a maltose feed. The pH was controlledusing 5N H₃PO₄ and 15% NH₄OH and maintained at 5.0 and then raised to5.25. Temperature was maintained 34.0° C.+/−1.0° C. Agitation was 1000rpm. Airflow was 1.0 vvm.

A 200 ml volume of cell-free supernatant was diluted to 1 liter withdeionized water. The pH was adjusted to 8 and the sample filtersterilized using a 0.22 μm polyethersulphone (PES) filter. The filtersterilized sample was loaded onto a 250 ml Q SEPHAROSE™ Fast Flow column(GE Healthcare, Piscataway, N.J., USA) pre-equilibrated with 25 mM TrispH 8. The enzyme was eluted from the column with a 0 to 1 M NaOHgradient in the same buffer. Fractions containing endoglucanase werepooled (400 ml) and the enzyme concentration calculated from thetheoretic extinction coefficient and the absorbance of the sample at 280nm.

Example 8: Preparation of Aspergillus fumigatus NN055679 Cel3ABeta-Glucosidase

Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ ID NO: 21[DNA sequence] and SEQ ID NO: 22 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2005/047499 using Trichodermareesei RutC30 as a host.

The broth was filtered using a 0.22 μm EXPRESS™ Plus Membrane. Thefiltered broth was concentrated and buffer exchanged using a tangentialflow concentrator (Pall Filtron, Northborough, Mass., USA) equipped witha 10 kDa polyethersulfone membrane (Pall Filtron, Northborough, Mass.,USA) and 20 mM Tris-HCl pH 8.5. The sample was loaded onto a QSEPHAROSE® High Performance column (GE Healthcare, Piscataway, N.J.,USA) equilibrated with 20 mM Tris pH 8.0, and bound proteins were elutedwith a linear gradient from 0-600 mM sodium chloride. The fractions wereconcentrated and loaded onto a SUPERDEX® 75 HR 26/60 column GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-150 mMsodium chloride pH 8.5. Protein concentration was determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 9: Preparation of Aspergillus fumigatus NN055679 GH10 Xylanase

Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (SEQ ID NO: 23 [DNAsequence] and SEQ ID NO: 24 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2as a host.

The broth was filtered using a 0.22 μm EXPRESS™ Plus Membrane. A 100 mlvolume of filtered broth was buffer exchanged into 50 mM sodium acetatepH 5.0 using a 400 ml SEPHADEX® G-25 column according to themanufacturer's instructions. Protein concentration was determined usinga Microplate BCA™ Protein Assay Kit with bovine serum albumin as aprotein standard.

Example 10: Preparation of Talaromyces emersonii CBS 393.64 GH3Beta-Xylosidase

Talaromyces emersonii CBS 393.64 (NN005049) beta-xylosidase (SEQ ID NO:25 [DNA sequence] and SEQ ID NO: 26 [deduced amino acid sequence]) wasprepared recombinantly according to Rasmussen et al., 2006,Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzaeJaL355 (WO 2003/070956) as a host.

The broth was filtered using a 0.22 μm EXPRESS™ Plus Membrane. A 100 mlvolume of filtered broth was buffer exchanged into 50 mM sodium acetatepH 5.0 using a 400 ml SEPHADEX™ G-25 column according to themanufacturer's instructions. Protein concentration was determined usinga Microplate BCA™ Protein Assay Kit with bovine serum albumin as aprotein standard.

Example 11: Preparation of Trichoderma reesei GH61B Polypeptide HavingCellulolytic Enhancing Activity

Trichoderma reesei GH61B polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 27 [DNA sequence] and SEQ ID NO: 28 [deduced aminoacid sequence]) was prepared recombinantly according to WO 2007/089290.

The broth containing the Trichoderma reesei GH61B polypeptide wasfiltered using a 0.22 μm EXPRESS™ Plus Membrane. The filtered broth wasconcentrated and buffer exchanged using a tangential flow concentrator(Pall Filtron, Northborough, Mass., USA) equipped with a 10 kDapolyethersulfone membrane (Pall Filtron, Northborough, Mass., USA) and20 mM Tris-HCl pH 8.0 and then purified using a Mono Q® HR 16/10 ionexchange chromatography column (GE Healthcare, Piscataway, N.J., USA) in20 mM Tris-HCl pH 8, using a linear 0 to 1 M NaCl gradient. Fractionscontaining the GH61B polypeptide were pooled based SDS-PAGE analysisusing a 8-16% CRITERION® Stain-Free SDS-PAGE gel (Bio-Rad Laboratories,Inc., Hercules, Calif., USA). The pool was further purified using aHILOAD® 26/60 SUPERDEX® 75 size exclusion chromatography column (GEHealthcare, Piscataway, N.J., USA) in 20 mM Tris-HCl pH 8.0 containing150 mM NaCl. Fractions containing the GH61B polypeptide were pooledbased on SDS-PAGE. Protein concentration was determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 12: Effect of GH61 Polypeptides Having Cellulolytic EnhancingActivity on PCS-Hydrolyzing Activity of a Trichoderma reesei-BasedCellulase Composition and a High-Temperature Enzyme Composition

Two GH61 polypeptides having cellulolytic enhancing activity,Trichoderma reesei GH61A and Trichoderma reesei GH61B, were eachevaluated for their ability to enhance the PCS-hydrolyzing activity of aTrichoderma reesei-based cellulase composition [a blend of 95%CELLUCLAST™ 1.5 L FG (Novozymes A/S, Bagsvaerd, Denmark) and 5%Aspergillus fumigatus beta-glucosidase based on protein] and ahigh-temperature enzyme composition (HT composition) using milledunwashed PCS at 50° C., 55° C., 60° C., and 65° C. Each GH61 polypeptidewas separately added at 0.525 mg of enzyme protein per gram cellulose tothe Trichoderma reesei-based cellulase composition (2.975 mg of enzymeprotein per gram cellulose) or the high-temperature enzyme composition(2.975 mg of enzyme protein per gram cellulose). The high-temperatureenzyme composition included 43.5% Penicillium emersonii Cel7Acellobiohydrolase I, 29.4% Aspergillus fumigatus Cel6A cellobiohydrolaseII, 11.8% Thermoascus aurantiacus Cel5A endoglucanase II, 5.9%Aspergillus fumigatus Cel3A beta-glucosidase, 5.9% Aspergillus fumigatusGH10 xylanase, and 3.5% Talaromyces emersonii GH3 beta-xylosidase. Theresults were compared with the results from the Trichoderma reesei-basedcellulase composition without the GH61 polypeptides having cellulolyticenhancing activity or the high-temperature enzyme composition withoutthe GH61 polypeptides having cellulolytic enhancing activity.

The assay was performed as described in Example 4. The 1 ml reactionswith 5% milled unwashed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results shown in FIG. 3 demonstrated that at 50° C. and 55° C. theTrichoderma reesei GH61A polypeptide and the Trichoderma reesei GH61Bpolypeptide were able to enhance the 72 hour PCS hydrolysis by theTrichoderma reesei-based cellulase composition. At 50° C. and 55° C.,the Trichoderma reesei GH61A polypeptide enhanced the 72 hour PCShydrolysis by the Trichoderma reesei-based cellulase composition to agreater extent than the Trichoderma reesei GH61B polypeptide. Theoptimal enhancement by the Trichoderma reesei GH61A polypeptide and theTrichoderma reesei GH61B polypeptide when added to the Trichodermareesei-based cellulase composition was at 50° C. and decreasedsignificantly at 55° C., 60° C., and 65° C. At 60° C. and 65° C., theaddition of the Trichoderma reesei GH61A polypeptide or the Trichodermareesei GH61B polypeptide to the Trichoderma reesei-based cellulasecomposition yielded minimal improvement of the 72 hour PCS hydrolysiscompared to the Trichoderma reesei-based cellulase composition at 2.975mg protein per g cellulose.

When both GH61 polypeptides were added to the high-temperature enzymecomposition, both showed cellulase-enhancing activity at 50° C. and 55°C. However, the Trichoderma reesei GH61A polypeptide showedsignificantly higher cellulase-enhancing activity at 50° C. and 55° C.compared to Trichoderma reesei GH61B polypeptide. While the Trichodermareesei GH61B polypeptide showed some enhancement at 50° C. and 55° C.,little or no enhancement was observed at 60° C. and 65° C. while theTrichoderma reesei GH61A polypeptide showed significantcellulase-enhancing activity at 60° C. and 65° C. when added to thehigh-temperature enzyme composition. Overall, the Trichoderma reeseiGH61A polypeptide showed significant enhancing performance across alltemperatures from 50-65° C. when added to the high-temperature enzymecomposition.

Viikari et al., 2007, supra, disclose that the Trichoderma reeseicellulase system is rapidly inactivated at temperatures above 45° C.,and the optimal temperature of the Trichoderma reesei cellulase systemis generally considered to be below 45° C. on lignocellulose substratesrequiring longer hydrolysis times. Moreover, Viikari et al., 2007,supra, disclose that only the Trichoderma reesei GH45A endoglucanase wassomewhat more resistant to thermal inactivation and retained mostactivity at higher temperatures, while GH12A and GH61A, as well asxylanases and other accessory enzymes, were inactivated.

The strong performance of the T. reesei GH61A polypeptide in thetemperature range 55° C. to 65° C. when added to a high-temperaturecellulolytic enzyme composition in a 72 hour hydrolysis of PCS wasunexpected considering that the temperature optimum of the Trichodermareesei cellulase system is below 45° C. for a 72 hour hydrolysisaccording to Viikari et al. The results shown herein demonstrated thatthe Trichoderma reesei-based cellulase composition performed poorlyabove 50° C. whether or not the T. reesei GH61A polypeptide was added tothe composition. Furthermore, the performance of the T. reesei GH61Bpolypeptide was poor in the temperature range 55° C. to 65° C. comparedto 50° C. when added to either the Trichoderma reesei-based cellulasecomposition or the high-temperature cellulolytic enzyme composition in a72 hour hydrolysis of PCS. Consequently, as the results demonstrateherein, the T. reesei GH61A polypeptide showed surprisingcellulase-enhancing activity when added to a high-temperature enzymecomposition at all temperatures particularly in the range of 55° C. to65° C. in a 72 hour hydrolysis of PCS. Based on the low temperatureoptimum of the Trichoderma reesei cellulase system, the results ofViikari et al., and the poor performance of the T. reesei GH61Bpolypeptide, it was surprising that the T. reesei GH61A polypeptideexhibited significant cellulase-enhancing activity at high temperatures.

The present invention is further described by the following numberedparagraphs:

[1] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material under high temperatureconditions with an enzyme composition in the presence of a GH61polypeptide having cellulolytic enhancing activity selected from thegroup consisting of: (a) a GH61 polypeptide having at least 60% sequenceidentity to the mature polypeptide of SEQ ID NO: 2; (b) a GH61polypeptide encoded by a polynucleotide that hybridizes under at leastmedium-high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii); (c) a GH61 polypeptide encoded bya polynucleotide having at least 60% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof; (d) a variant of the mature polypeptide of SEQ ID NO: 2comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and (e) a fragment of the GH61 polypeptide of(a), (b), (c), or (d) that has cellulolytic enhancing activity.

[2] The method of paragraph 1, wherein the GH61 polypeptide has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the mature polypeptide of SEQ ID NO: 2.

[3] The method of paragraph 1 or 2, wherein the GH61 polypeptide isencoded by a polynucleotide that hybridizes under medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii).

[4] The method of any of paragraphs 1-3, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

[5] The method of any of paragraphs 1-4, wherein the GH61 polypeptidecomprises or consists of SEQ ID NO: 2.

[6] The method of any of paragraphs 1-4, wherein the GH61 polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 2.

[7] The method of paragraph 6, wherein the mature polypeptide is aminoacids 22 to 344 of SEQ ID NO: 2.

[8] The method of any of paragraphs 1-4, wherein the GH61 polypeptide isa variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions.

[9] The method of any of paragraphs 1-4, wherein the GH61 polypeptide isa fragment of SEQ ID NO: 2, wherein the fragment has cellulolyticenhancing activity.

[10] The method of any of paragraphs 1-9, wherein the cellulosicmaterial is pretreated.

[11] The method of any of paragraphs 1-10, wherein the enzymecomposition comprises one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[12] The method of paragraph 11, wherein the cellulase is one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[13] The method of paragraph 11, wherein the hemicellulase is one ormore (e.g., several) enzymes selected from the group consisting of axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[14] The method of any of paragraphs 1-13, further comprising recoveringthe degraded cellulosic material.

[15] The method of paragraph 14, wherein the degraded cellulosicmaterial is a sugar.

[16] The method of paragraph 15, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[17] The method of any of paragraphs 1-16, wherein the enzymecomposition and/or the GH61 polypeptide having cellulolytic enhancingactivity are in the form of a fermentation broth with or without cells.

[18] The method of any of paragraphs 1-17, wherein the high temperatureconditions are a temperature of about 54° C. to about 70° C. for about 6to about 168 hours at a pH of about 3 to about 8 and a dry solidscontent of a cellulosic material of about 5 to about 50 wt %.

[19] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material under high temperature conditionswith an enzyme composition in the presence of a GH61 polypeptide havingcellulolytic enhancing activity; (b) fermenting the saccharifiedcellulosic material with one or more (e.g., several) fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation; wherein the GH61polypeptide having cellulolytic enhancing activity is selected from thegroup consisting of: (a) a GH61 polypeptide having at least 60% sequenceidentity to the mature polypeptide of SEQ ID NO: 2; (b) a GH61polypeptide encoded by a polynucleotide that hybridizes under at leastmedium-high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii); (c) a GH61 polypeptide encoded bya polynucleotide having at least 60% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof; (d) a variant of the mature polypeptide of SEQ ID NO: 2comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions; and (e) a fragment of the GH61 polypeptide of(a), (b), (c), or (d) that has cellulolytic enhancing activity.

[20] The method of paragraph 19, wherein the GH61 polypeptide has atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 2.

[21] The method of paragraph 19 or 20, wherein the GH61 polypeptide isencoded by a polynucleotide that hybridizes under medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii).

[22] The method of any of paragraphs 19-21, wherein the GH61 polypeptideis encoded by a polynucleotide having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.

[23] The method of any of paragraphs 19-22, wherein the GH61 polypeptidecomprises or consists of SEQ ID NO: 2.

[24] The method of any of paragraphs 19-22, wherein the GH61 polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 2.

[25] The method of paragraph 24, wherein the mature polypeptide is aminoacids 22 to 344 of SEQ ID NO: 2.

[26] The method of any of paragraphs 19-22, wherein the GH61 polypeptideis a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions.

[27] The method of any of paragraphs 19-22, wherein the GH61 polypeptideis a fragment of SEQ ID NO: 2, wherein the fragment has cellulolyticenhancing activity.

[28] The method of any of paragraphs 19-27, wherein the cellulosicmaterial is pretreated.

[29] The method of any of paragraphs 19-28, wherein the enzymecomposition comprises one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[30] The method of paragraph 29, wherein the cellulase is one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[31] The method of paragraph 29, wherein the hemicellulase is one ormore (e.g., several) enzymes selected from the group consisting of axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[32] The method of any of paragraphs 19-31, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[33] The method of any of paragraphs 19-32, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, analkane, a cycloalkane, an alkene, isoprene, polyketide, or a gas.

[34] The method of any of paragraphs 19-33, wherein the enzymecomposition and/or the GH61 polypeptide having cellulolytic enhancingactivity are in the form of a fermentation broth with or without cells.

[35] The method of any of paragraphs 19-34, wherein the high temperatureconditions are a temperature of about 54° C. to about 70° C. for about 6to about 168 hours at a pH of about 3 to about 8 and a dry solidscontent of a cellulosic material of about 5 to about 50 wt %.

[36] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more (e.g., several)fermenting microorganisms, wherein the cellulosic material issaccharified under high temperature conditions with an enzymecomposition in the presence of a GH61 polypeptide having cellulolyticenhancing activity selected from the group consisting of: (a) a GH61polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii); (c) a GH61 polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof; (d) a variant ofthe mature polypeptide of SEQ ID NO: 2 comprising a substitution,deletion, and/or insertion at one or more (e.g., several) positions; and(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

[37] The method of paragraph 36, wherein the GH61 polypeptide has atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 2.

[38] The method of paragraph 36 or 37, wherein the GH61 polypeptide isencoded by a polynucleotide that hybridizes under medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii).

[39] The method of any of paragraphs 36-38, wherein the GH61 polypeptideis encoded by a polynucleotide having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.

[40] The method of any of paragraphs 36-39, wherein the GH61 polypeptidecomprises or consists of SEQ ID NO: 2.

[41] The method of any of paragraphs 36-39, wherein the GH61 polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 2.

[42] The method of paragraph 41, wherein the mature polypeptide is aminoacids 22 to 344 of SEQ ID NO: 2.

[43] The method of any of paragraphs 36-39, wherein the GH61 polypeptideis a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions.

[44] The method of any of paragraphs 36-39, wherein the GH61 polypeptideis a fragment of SEQ ID NO: 2, wherein the fragment has cellulolyticenhancing activity.

[45] The method of any of paragraphs 36-44, wherein the cellulosicmaterial is pretreated before saccharification.

[46] The method of any of paragraphs 36-45, wherein the enzymecomposition comprises one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[47] The method of paragraph 46, wherein the cellulase is one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[48] The method of paragraph 46, wherein the hemicellulase is one ormore (e.g., several) enzymes selected from the group consisting of axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[49] The method of any of paragraphs 36-48, wherein the fermenting ofthe cellulosic material produces a fermentation product.

[50] The method of paragraph 49, further comprising recovering thefermentation product from the fermentation.

[51] The method of paragraph 49 or 50, wherein the fermentation productis an alcohol, an organic acid, a ketone, an amino acid, an alkane, acycloalkane, an alkene, isoprene, polyketide, or a gas.

[52] The method of any of paragraphs 36-51, wherein the enzymecomposition and/or the GH61 polypeptide having cellulolytic enhancingactivity are in the form of a fermentation broth with or without cells.

[53] The method of any of paragraphs 36-52, wherein the high temperatureconditions are a temperature of about 54° C. to about 70° C. for about 6to about 168 hours at a pH of about 3 to about 8 and a dry solidscontent of a cellulosic material of about 5 to about 50 wt %.

[54] A detergent composition comprising a GH61 polypeptide havingcellulolytic enhancing activity selected from the group consisting of:(a) a GH61 polypeptide having at least 60% sequence identity to themature polypeptide of SEQ ID NO: 2; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii); (c) a GH61 polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof; (d) a variant ofthe mature polypeptide of SEQ ID NO: 2 comprising a substitution,deletion, and/or insertion at one or more (e.g., several) positions; and(e) a fragment of the GH61 polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

[55] The composition of paragraph 54, further comprising one or more(e.g., several) of a cellulase, a protease, a lipase, a cutinase, anamylase, a carbohydrase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase.

[56] The composition of paragraph 54 or 55, which is formulated as abar, a tablet, a powder, a granule, a paste or a liquid.

[57] A method for cleaning or washing a hard surface or laundry, themethod comprising contacting the hard surface or the laundry with thecomposition of any of paragraphs 54-56.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-20. (canceled)
 21. A method of fermenting a cellulosic material,comprising: fermenting the cellulosic material with one or morefermenting microorganisms, wherein the cellulosic material issaccharified under high temperature conditions with an enzymecomposition in the presence of a GH61 polypeptide having cellulolyticenhancing activity selected from the group consisting of: (a) a GH61polypeptide having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 2; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii),wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) a GH61polypeptide encoded by a polynucleotide having at least 95% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof; and (d) a fragment of the GH61 polypeptide of(a), (b), or (c) that has cellulolytic enhancing activity; wherein thehigh temperature conditions are a temperature of about 54° C. to about70° C. for about 6 to about 168 hours at a pH of about 3 to about 8 anda dry solids content of a cellulosic material of about 5 to about 50 wt%; wherein an effective amount of the GH61 polypeptide is about 0.01 toabout 50.0 mg per g of the cellulosic material.
 22. The method of claim21, wherein the GH61 polypeptide has at least 96% sequence identity tothe mature polypeptide of SEQ ID NO:
 2. 23. The method of claim 21,wherein the GH61 polypeptide has at least 97% sequence identity to themature polypeptide of SEQ ID NO:
 2. 24. The method of claim 21, whereinthe GH61 polypeptide has at least 98% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 25. The method of claim 21, wherein theGH61 polypeptide has at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 26. The method of claim 21, wherein theGH61 polypeptide comprises SEQ ID NO: 2 or the mature polypeptidethereof.
 27. The method of claim 21, wherein the GH61 polypeptideconsists of the mature polypeptide of SEQ ID NO:
 2. 28. The method ofclaim 21, wherein the GH61 polypeptide is encoded by a polynucleotidethat hybridizes under very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence thereof, or (iii) the full-length complement of (i) or (ii),wherein very high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 50% formamide, and washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.
 29. Themethod of claim 21, wherein the GH61 polypeptide is encoded by apolynucleotide having at least 96% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.
 30. The method of claim 21, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 97% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.
 31. The method of claim 21, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 98% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.
 32. The method of claim 21, wherein the GH61 polypeptide isencoded by a polynucleotide having at least 99% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.
 33. The method of claim 21, wherein the GH61 polypeptide isencoded by a polynucleotide comprising the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof.
 34. The method ofclaim 21, wherein the GH61 polypeptide is encoded by a polynucleotideconsisting of the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof.
 35. The method of claim 21, wherein thecellulolytic enzyme composition further comprises one or more enzymesselected from the group consisting of a cellulase, a hemicellulase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.
 36. The method of claim 35,wherein the cellulase is one or more enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase.
 37. The method of claim 35, wherein the hemicellulaseis one or more enzymes selected from the group consisting of a xylanase,an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.
 38. The method of claim 21, wherein thecellulosic material is pretreated.
 39. The method of claim 21, whereinthe fermenting of the cellulosic material produces a fermentationproduct.
 40. The method of claim 39, further comprising recovering thefermentation product from the fermentation.
 41. The method of claim 40,wherein the fermentation product is an alcohol, an organic acid, aketone, an amino acid, an alkane, a cycloalkane, an alkene, isoprene,polyketide, or a gas.
 42. The method of claim 21, wherein thecellulolytic enzyme composition and/or the GH61 polypeptide havingcellulolytic enhancing activity are in the form of a fermentation brothwith or without cells.